The values of charge and electrophoretic mobility of a protein are changed upon acylation of its alpha- and Lys epsilon-NH3+ groups. Partial acylation of the amino groups of a protein results in a set of derivatives that is often resolved by capillary electrophoresis into a set of distinct peaks-the "rungs" of a protein charge ladder-that differ incrementally in the number of residues modified. Proteins that have values of MW < 50 kD usually form resolved charge ladders when allowed to react with acetic anhydride, while proteins that have values of MW > 50 kD form broad unresolved peaks. Resolved charge ladders of proteins that have values of MW > 50 kD may be formed using acylating agents that introduce several charges upon acylation of each of their Lys epsilon-NH3+ groups. As an example, L-lactate dehydrogenase (MW = 147 kD) does not form a resolved charge ladder when modified with acetic anhydride. When it is acylated with either 1,2,4-benzenetricarboxylic anhydride, 3, or 1,2,4,5-benzenetetracarboxylic dianhydride, 4, however, it forms charge ladders in which each of the first several pairs of adjacent rungs are separated by approximately 3 or 4 units of charge, respectively. The procedures described in this paper were used to form resolved charge ladders from 25 proteins differing in pI and in MW.